**1. Introduction**

The genus *Leishmania* belongs to the order Trypanosomatida and includes protozoan parasites that are responsible for a complex of diseases named leishmaniasis, which is the second most common cause of mortality among tropical infectious diseases, after malaria [1]. Some species of *Leishmania* are human pathogens that cause different clinical manifestations as cutaneous (CL), mucocutaneous (MCL), or visceral (VL) leishmaniasis. Old World species *Leishmania infantum* and *Leishmania donovani* cause VL or kala-azar, which is often lethal if untreated, whereas *Leishmania major* causes CL and *Leishmania braziliensis* is associated with MCL. The VL-causative species are genetically almost identical, although they differ in geographic distribution: *L. donovani* is found in the Indian subcontinent and East Africa, while *L. infantum* is endemic in the countries around the Mediterranean basin, Latin America, and China [2]. Two stages, promastigote and amastigote, alternate in the *Leishmania* life cycle. Promastigotes are flagellated and motile forms develop extracellularly in the gut of their sand-fly

vector. The infection of the mammal host takes place during the sand-fly blood meal; afterwards, parasites are phagocytized by macrophages, and amastigote forms develop inside these host cells.

Within the *Leishmania* genus, the *L. major* genome was the first to be sequenced [3], followed by the *L. infantum* and *L. braziliensis* ones [4]. During the last decade, the extraordinary progress in DNA sequencing methodologies has allowed for the drafting of the genomes for many other *Leishmania* species [5–12] and for the improvement of the assemblies of the first sequenced genomes [13–15].

The availability of a complete and well-annotated genome provides the ultimate resource for genome-wide scale approaches, such as transcriptome and proteome analyses [16]. In parallel to the advances in sequencing technologies, proteomics methodologies are achieving unprecedented levels of sensitivity, and novel MS-based experimental approaches have become the method of choice for the analysis of complex protein mixtures such as cells, tissues, and even whole organisms. In particular, several proteomic technologies are being used to study diverse aspects of *Leishmania* biology such as parasite development, virulence, and drug resistance [17]. Thus, proteomics approaches have been used to determine differential patterns of protein expression between the promastigote and amastigote stages in *Leishmania mexicana* [18], *L. infantum* [19], and *L. donovani* [20], among others. Other studies have been aimed to ascertain specific proteomes by means of organelle fractionation to obtain enriched fractions of mitochondria, flagella, or glycosomes [21,22]. The identification and mapping of protein post-translational modifications (PTMs) provide additional information about the activation of specific pathways in a given growing condition, thus improving the knowledge on protein interactions in complex networks.

Here, we present a wide and detailed proteome of the *L. infantum* JPCM5 strain, based on axenically grown promastigotes in the logarithmic growth phase. A careful comparative analysis with other published proteomes from different Old and New World *Leishmania* species has also been carried out. Additionally, the MS data allowed for the identification of PTMs (phosphorylation, acetylation, methylation, formylation, and glycosylation) at specific protein sites that might have regulatory functions. Furthermore, we showed how the integration of proteomics with genomic and transcriptomic data represents a powerful and complementary strategy for gene annotation, as demonstrated before in a plethora of species [23]. Hence, by applying this proteogenomic approach, it was possible to improve the annotations for several *L. infantum* genes, as well as the identification of eight novel genes.
